JP2803336B2 - Plant tissue culture method - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a method for producing a medium without using an autoclave, and a method for culturing a plant tissue which can achieve the same effect as an aseptic operation without using a clean bench.

2. Description of the Related Art Generally, when performing tissue culture of a plant, a plant tissue is grown under sterile conditions using a medium containing nutrients necessary for plant growth. The reason for this is that when the inside of the culture vessel is contaminated with fungi, the plants do not grow because the growth of the fungi is faster than the growth of the plants. Therefore, conventionally, the mainstream of the plant tissue culturing method has been to use a medium sterilized using an autoclave and perform the operation under aseptic conditions with a clean bench operated.

An autoclave is a device similar to the principle of a pressure cooker, in which a medium placed in a pressure vessel is treated at high temperature and high pressure to kill microorganisms in the medium, and a clean bench supplies sterile air filtered. Device.

Problems to be Solved by the Invention Both the autoclave and the clean bench are large and expensive devices. Autoclaves require a long time and sterilization due to the high temperature and high pressure inside the autoclave. The clean bench takes time to make the operating environment aseptic, and even after the aseptic condition, it must be operated with great care so as not to be contaminated by various bacteria.

An object of the present invention is to solve the above-mentioned problems, and an object of the present invention is to provide a culture method that is not contaminated by fungi without using an autoclave and a clean bench.

Means for solving the problem The present invention, in order to achieve the above object, is a pungent component of wasabi, antiallyl allyl isothiocyanate,
The present invention is characterized in that tissue culture is performed in a culture vessel. There are three specific methods for implementing this.

A method in which allyl isothiocyanate is added to a medium when the medium is prepared.

A method of spraying on the surface of a medium in allyl isothiocyanate during culture.

A method in which allyl isothiocyanate is applied to the culture vessel wall during culture.

Effect When the above-described method is used, aseptic operation is not required, so that tissue culture of a plant can be performed easily and at low cost.

Allyl isothiocyanate is the major pungent component of wasabi. Although it has been known that this substance has an antibacterial effect, its use for plant tissue culture has not been reported to date. The inventor of the present application attempted to use allyl isothiocyanate for the purpose of simplifying the operation and reducing the cost when performing tissue culture of a plant, and by adding this chemical, the same method was performed without performing a conventional aseptic operation. For the first time, we have found that it works.

Embodiment Hereinafter, an embodiment of the present invention will be described with reference to the drawings.

Example 1 A case of preparing a medium will be described with reference to FIG. As a basic operation, the medium is adjusted in advance according to a conventional method.
Then, when heated to about 100 degrees and the gelling agent dissolves,
Allyl isothiocyanate diluted 100-fold with ethanol is added to prepare a medium. The addition volume is 0.1 ml to 1.0 ml per 30 ml of the medium. Next, the solution was dispensed into a culture vessel and covered with an aluminum foil, whereby the volatilized allyl isothiocyanate was filled in the vessel. After that, it was left as it was in a 25 ° C. incubator for one month, but aseptic colonies were not observed. Thus, it was confirmed that the internal bacteria were killed without using an autoclave.

After that, when the carnation plant that had been subcultured by the conventional tissue culture method was transplanted under a clean bench to the medium created here, one month had passed.
No bacterial contamination was found in the medium. No abnormalities were observed in the plant.

Example 2 The case of aseptic seeding will be described with reference to FIG. First, seeds of sweet pea were pre-treated with 70% ethanol for 5 minutes,
Sterilize sequentially with 20% sodium hypochlorite for 20 minutes. Next, at the time of seeding, the medium prepared in Example 1 is used, and the surface of the medium is further diluted 100-fold with ethanol and sprayed with allyl isothiocyanate. At this time, the spraying capacity is 100m
It is 0.1 ml to 1.0 ml for l. Thereafter, seeding was performed without using a clean bench, and the cells were covered with aluminum foil with a filter of 0.02 μm. Thereafter, the cells were cultured for 3 months, but no aseptic colonies were found even on the surface of the culture medium that was originally most likely to be contaminated. The vaporized allyl isothiocyanate was considered to be diffused out of the container through the filter, and no abnormalities were observed in the germinated plants.

Example 3 The case of subculture will be described with reference to FIG. Example 1
First, allyl isothiocyanate diluted 100-fold with ethanol was applied to the inside of the culture vessel without touching the medium, using the medium prepared in 1). The amount of application is 100ml flask
0.1 ml to 0.5 ml. Next, strawberry plants were transplanted and covered with a 0.02 μm aluminum foil with a filter.

Thereafter, the cells were cultured for one month, but no aseptic colonies were found. In this experiment, the additional applied allyl isothiocyanate was not mixed into the culture medium, so that the concentration of allyl isothiocyanate in the culture medium was not increased, and it is considered that the harm to the plants was further reduced. I couldn't see it. Also in this case, it is considered that allyl isothiocyanate vaporized in the same manner as in Example 2 is diffused outside the container through the filter.

In addition, in Examples 2 and 3 in which abnormalities were observed, all the mediums were supplemented with allyl isothiocyanate, but instead, an autoclave may be used only for sterilization of the medium. In this case, autoclaving would be required, but the harm to the plant would be even less.

Example 4 A case of liquid culture will be described with reference to FIG. As a basic operation, a liquid medium is prepared in advance according to an ordinary method, and allyl isothiocyanate diluted 100-fold with ethanol is added to prepare a medium. The addition volume is 30 ml of medium,
0.1 ml to 1.0 ml. Next, after dispensing, it was covered with aluminum foil. After standing in a 25 ° C. incubator for one month at room temperature, no turbidity was observed in the culture solution.
Therefore, it is considered that by filling the container with allyl isothiocyanate, the bacteria inside can be killed without using an autoclave. Thereafter, when the protocorm-like body of cymbidium was further transplanted to this medium in the same manner as in Example 3, even after 3 months, the cells grew well and no turbidity of the culture was observed.

In all of the above examples, the instruments used for the operation were not sterilized.

In addition to the above examples, orchid family, asteraceae, primrose family, lily family, rose family, gramineae family, solanaceae family, seriaceae, legume family, crucifer family, cucurbitaceae family, citrus family, purple family, taro family, Amaryllidaceae, Iridaceae, Papilionidae, Cactiaceae, Salixaceae, Fagaceae, Camellia family, Malvaceae, Poppyceae, Rubiaceae, Polygonaceae, Palmae, Euphorbiaceae, but similar experiments were performed. The effect of was confirmed.

According to the method of the present invention, the antibacterial activity of allyl isothiocyanate is effectively utilized without performing a normal aseptic operation,
Tissue culture of a plant can be performed with a simple method and at low cost, and the industrial effect is extremely large.

[Brief description of the drawings]

FIG. 1 is a conceptual view of a culture vessel showing a state in which a solid medium to which allyl isothiocyanate is added is placed. FIG. 2 is a conceptual view of a culture vessel showing a state in which allyl isothiocyanate is sprayed on the surface of the medium. FIG. 4 is a conceptual diagram of a culture vessel showing a state in which allyl isothiosoanoate is applied without touching the culture medium on the wall of the vessel. FIG. 4 is a conceptual view of a culture vessel showing a state in which a liquid culture medium containing allyl isothiocyanate is added. It is a conceptual diagram. 1 ... a solid medium containing allyl isothiocyanate, 2 ...
Culture vessel, 3 ... aluminum foil, 4 ... seeds of sweet pea, 5 ... allyl isothiocyanate diluted 100 times, 6 ... aluminum foil with filter of 0.02μ, 7
... Strawberry plant, 8 ... Liquid culture medium containing allyl isothiocyanate.

Claims (4)

(57) [Claims] 1. A method for culturing a plant tissue, wherein allyl isothiocyanate CH ₂ CHCHCH ₂ H ＝ C ＝ S is placed in a culture vessel and cultured. 2. The method according to claim 1, wherein allyl isothiocyanate is added to the medium when the medium is prepared. 3. The method according to claim 1, wherein allyl isothiocyanate is sprayed on the surface of the culture medium in the container during the culturing. 4. The method according to claim 1, wherein allyl isothiocyanate is applied to the inner wall of the culture vessel during the culture.
A tissue culture method for a plant according to the above.